Preclinical development of complex processing systems for ex vivo culture- expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, has been carried out in collaboration with a number of NIH investigators, and with Baxter Healthcare, Inc. A. Preparation of CD8-depleted, culture-expanded lymphocytes: In collaboration with Drs. Robert Walker, Michael Blaese, and Richard Morgan, this process involves CD8 cell depletion of peripheral blood lymphocytes using a Baxter antibody and Max Sep, an immunomagnetic system for large- scale negative selection, prior to ex vivo culture-expansion and gene transduction. To date, there have been 19 large-scale runs (5 preclinical and 14 clinical) for this 10-day process, which is currently being applied to normal syngeneic donor lymphocytes in an HIV clinical gene therapy trial. This trial is ongoing and beginning to yield important data on the relative efficacy of a therapeutic gene vs a marker gene. Process changes this year were based on studies of paramagnetic bead concentrations for the CD8-negative selection. B. Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T cells: This process is being developed, in collaboration with Dr. John Barrett and colleagues, for application to clinical allogeneic hematopoietic transplantation, especially in the HLA-mismatched setting. It involves (1) preparation of recipient (stimulator) lymphocytes, (2) preparation of donor (responder) lymphocytes, and (3) co- culture and preparation of stimulator and responder lymphocytes, followed by selective depletion of alloreactive responder T lymphocytes with an anti-CD25-Pseudomonas exotoxin construct. This year we have concentrated on developing culture-expansion methods that will eliminate leukemia cells from the normal recipient (stimulator) cells, including methods for culture-expansion of selected lymphocyte populations. Further studies will continue into next year, as we prepare for a clinical trial.